Our goal is to elucidate the physical and molecular basis of homeostasis and misfolded protein stress in the endoplasmic reticulum (ER). Correct folding and quality control of secretory proteins by ER chaperones is essential for the viability of cells and organisms. Failure to correctly fold proteins results in loss of protein function, can activate the Unfolded Protein Response (UPR), and stimulate apoptotic death. Whether chaperones are sufficiently available for nascent or misfolded proteins under different environmental conditions is critical to understanding chaperone function. The ER chaperone network at steady state could be buffered with redundancy and an excess of chaperones or the network could be operating at the edge of protein folding capacity. The nature of the ER chaperone network has deep implications for how the ER senses and copes with misfolded protein stress. A system operating at capacity leaves little room for error, will have an explicit threshold for stress, and is likely to exhibit biphasic extremes. In contrast, stress and homeostasis could exist as a gradient of states in a buffered and redundant system. Small perturbations in folding requirements could be sensed and responded to, without upsetting the global folding environment. We hypothesize that the complexity and buffering capacity of the ER chaperone network regulates activation of ER stress pathways and maintenance of ER homeostasis. Chaperone network complexity will depend on the distribution, dynamics, organization, and occupancy of lumenal ER chaperones. While genetics and test-tube biochemistry have helped define the folding activities of chaperones, it remains poorly understood how ER chaperones encounter and interact with their substrates in cells in real time. We will employ biochemical, pharmacologic, and quantitative single cell fluorescence microscopy methods to establish a model of the ER chaperone network with exquisite spatio-temporal resolution. Our biophysical systems approach will define the hierarchy of the ER chaperone network during homeostasis and misfolded protein stress. PUBLIC HEALTH RELEVANCE The processes of correct secretory protein folding and quality control are vital for cell viability and human health. We are studying, at the cellular level, the mechanisms that maintain and regulate the secretory protein folding environment.